Cell and vaccine production

ABSTRACT

Use of tumbling in a rotating disc propagator to evenly distribute the cell slurry, obtain two side plating of cells, increase the cell concentration and recover the cells from the propagator in small volumes of medium, whereby the overall yield of cells and vaccines is significantly increased and at substantially reduced costs as compared to presently utilized procedures.

[ 1 Oct. 1, 1974 United States Patent McAleer et al.

3,407,120 lO/l968 Weiss et l95/l43 3,732,l49

[ CELL AND VACCINE PRODUCTION 5/l973Santero......,.................,...... l95/l27 [75] Inventors: WilliamJ. McAleer, Ambler;

Raymond E. Spier; Kenneth L.

a Wt 0N V Mb 3 mR m. m l 0- 0 MC b& & r Me PM a e n .m S S A i 3 r i[22] Filed: July 27, 1972 ABSTRACT 21 Appl. No.: 275,642

Use of tumbling in a rotating disc propagator to evenly [52] 195/127distribute the cell slurry, obtain two side plating of cells, increasethe cell concentration and recover the cells from the propagator insmall volumes of medium, whereby the overall yield of cells and vaccinesis significantly increased and at substanti 03 04 1.. l b2 24 1 9 m3 .l2 NH 5 9 Mb c r a e S 1 m d Ln .mm.. ii] 8 55 [i ally reduced costs ascompared to presently utilized procedures.

3 Claims, 6 Drawing Figures 2,996,429 Toulmin, Jr. 195/143 CELL ANDVACCINE PRODUCTION This invention relates to the production of cells andvaccines.

More particularly, this invention relates to the use of tumbling inrotating disc machines in order to produce cells and vaccines insubstantially increased yields, thereby greatly reducing productioncosts.

Human and animal vaccines have been commercially produced by growing thedesired virus in primary cells which must be grown on surfaces.Commerical processes were initially developed in Brockway bottles. Theseprocesses required the use of thousands of individual bottles to achievethe production of sufficient quantities of vaccine. The use of such alarge number of bottles or production units is very time consuming andcostly, and creates a substantial risk of contamination. As productiontechniques evolved, the original Brockway bottles were replaced byroller bottles which only slightly reduced the number of bottles and thehandling problems associated therewith.

Some mass culture systems have been developed, such as the multiplateunit disclosed in U.S. Pat. No. 3,407,120 and the Biotec cylindricalrotating disc apparatus, but these units provide only minimal advantagesover the original individual bottle system. In using a rotating discapparatus, the greatest efficiency is obtained by minimizing the spacebetween the plates, and growing cells on both sides of the plates. Thishowever, creates a serious problem in obtaining a uniform distributionof cells and/or virus over both planar surfaces of the plates.

The present invention provides a method and device for overcoming theaforementioned disadvantages of the prior art procedures in which acylindrical rotating disc apparatus is tumbled end over end atcontrolled speeds during the cell and vaccine production cycles, as forexample during the cell plating, cell growth, cell washing, virusseeding, virus replication, and harvesting cycles of operation or duringthe addition of a chemical agent to the growth unit. For example, duringthe cell plating cycle, the nropagator can be tumbled at a speed of fromabout 1 rev./5 min. to about 1 rev./l5 min., preferably at a speed ofabout 1 rev./l min., while during the virus seeding cycle, thepropagator can be tumbled at a speed of from about 1 rev./3 min. toabout I rev./8 min., preferably at a speed of l rev./ min. and duringthe harvesting cycle at a speed of from about 30 rev./min. to about 90rev./min., preferably at a speed of about 60 rev./min.

An advantage of the present invention is the ability to substantiallyincrease the yield of cells and vaccines and thereby reduce theproduction costs.

A further advantage of the present invention is the ability to evenlydistribute the cell slurry between the plates, thereby obtaining greateruniformity in the cell distribution and a higher yield of cells andvaccine.

A still further advantage of the present invention is the ability toobtain plating of cells on both sides of the plates from a single chargeof cells without the necessity ofa two-step procedure which requires twoseparate charges of cells, thereby increasing the yield of cells andreducing the risk of contamination and the costs associated therewith.

Another advantage of the present invention is that extremely low volumesof fluid can be used to bathe the cell sheet, and to recover the cellsfrom the tank,

thereby increasing the yield of cells or the titre/ml. of cellassociated vaccine when compared to prior art procedures.

These and other advantages of the present invention will be readilyunderstood by reference to the following detailed description whenconsidered in conjunction with the accompanying drawings in which:

FIG. 1 is a perspective view of a typical multiplate propagator andholding means for the propagator employed in the present invention;

FIG. 2 is a cross sectional view of the propagator contained in theholding means;

FIG. 3 is a sectional view taken along the line 33 of FIG. 2; I

FIG. 4 is a front elevational view of the tumbling device of the presentinvention;

FIG. 5 is a side elevational view of the tumbling device of the presentinvention; and

FIG. 6 is a sectional view of another propagator which is exemplary ofthose which can be utilized in accordance with the teachings of thisinvention.

Referring to the drawings, FIG. 1 discloses the multiplate propagator(l) and the holding means (2). The propagator (1) includes a cylindricalstainless steel vessel (3) having top and bottom flanges (4) and (5) anda top plate (6) and a bottom plate (7). Clamps (8) are used to sealplates (6) and (7) to flanges (4) and (5) respectively. Plates (6) and(7) also have fittings (9), (10), (11), and (12) which connect tovarious lines when the propagator is in use in order to cycle air andfluids through the propagator. The holding means (2) also shown in FIG.1 includes a cage like structure having two semi-tubular portions (13)and (14) which may be of an open or closed construction and areconnected by hinges (l5). Portions (13) and (14) each have flanges (16)and (17) which have holes (18) through which pins or clamps may beinserted to close and latch the holding means (2) around the propagator(l). The holding means (2) also has two cylindrical arms (19) and (20)extending from opposite sides of the midpoint of the holding means.

As illustrated in FIG. 2 the propagator (1) contains a series oftitanium discs (21) mounted on a bar (22), which supports the plates (7)in a separated state due to the presence of cylindrical spacers betweeneach plate. One end of the bar is rotatably supported by a bearing (23)which is mounted in a recess (24) in the center of plate (7). The otherend of the bar (22) is rotatably supported by a bearing (25) which ismounted in a recess (26) in the center of plate (6). A magnetic couple(25) which is fixedly mounted on the bar (20) is engaged by magneticdrive means (not shown) in order to rotate the plates. The holding means(2) envelops the propagator and the edges of the semi-tubular members(13) and (14) about the edges of flanges (4) and (5) in order to holdthe tank (1) in a fixed position.

FIG. 3 is a sectional view taken along line 33 of FIG. 2, and shows thetwo semi-tubular portions (13) and (14) in the closed position and a pin(28) inserted in the holes (18) in flanges (1 6) and (17) to lock theholding means in the closed position around the cylindrical portion (3)of the tank (1).

FIG. 4 is a front elevational view of the tumbling device of thisinvention and shows the propagator (1) contained in the holding means(2). Arm (19) is surrounded by a bearing (29) rotatably mounted in hole(30) of supporting member (31) and arm (20) is fixedly mounted insprocket wheel (32) which in turn is rotatably mounted on support member(33). Wheel (32) is connected to sprocket wheel (34) of a motor (35) bya chain (36) in order to tumble the propagator (l). The motor (35) has avariable speed transmission in order to tumble the propagator at thedesired speed. Support members (31) and (33) are positioned on stand(37).

FIG. is a side elevational view of the tumbling device of FIG. 4 andillustrates the manner in which the propagator is tumbled end over endwhile positioned in the tumbling device.

Similarly in FIG. 6, there is disclosed another multiplate propagator(51) which can be used in conjunction with the tumbling device of thisinvention. This propagator (51) comprises a cyclindrical stainless steelvessel (52) having a flange (53) at one end thereof. Plate (54) issealed to the flange (53) by clamps (55). An air-carbon dioxide mixturecan be pumped into the vessel (52) from a reservoir (not shown) througha line (56) which extends along the length of the wall of the vessel(52) to the back of the vessel (52) where a portion of the line (56)extends along the back of the vessel. This portion of the line (56) hasopenings (57) which permit the egress of the air-carbon dioxide mixture.An outlet line (58) is also used to keep the air pressure within thevessel at a relatively constant level. Another line (59) may be used tosupply medium, serum and other nutrients and to withdraw the expendedmedium and product. The plates (60) are mounted on a bar (61 whichsupports the plates (54) in a separated state due to the presence ofcylindrical spacers between each plate. One end of the bar (61) isrotatably supported by a bearing (62) which is mounted in recess (63) inthe bottom of the vessel (52). The other end of the bar (61) is alsorotatably supported by a bearing (64) which is mounted in a recess (65)in plate (54). A magnetic couple (66) which is fixedly mounted on bar(61) is engaged by a driven magnet (67) to rotate the plates (60)through the medium (58) during the cell growth and virus infectionstages of the production cycle.

The process and device of this invention may be used to produce viralvaccines such as mumps, measles, rubella, parainfluenza, Mareks andcells such as WI-38, chick embryo and duck embryo cells. Standard cells,sera and media may be used to produce the aforementioned vaccines. Forexample, primary cells such as chick embryo fibroblasts, green monkeykidney, bovine kidney, dog kidney or diploid cells such as WI-38 may beutilized as may standard sera such as fetal calf, calf, bovine, G-G-freenewborn calf, a-gamma calf or a-gamma bovine and standard media such asEagles Basel Medium, Medium EBME, Medium 199, and Eagles MinimumEssential Medium.

The invention will be better understood by reference to the followingexamples.

EXAMPLE 1 A rotating titanium disc propagator is charged with a mixtureof 12 billion trypsinized chick embryo cells in Medium 199,45 ml. 2.8%NaHCO /L and fetal calf serum. The charged propagator is then lockedinto the tumbler and tumbled at 37C. end over end at a speed of lrev./l0 min. until two-side plating has occurred at which time thepropagator is removed from the tumbler. The propagator is thenpositioned so that the plane of the discs is in the vertical axis and aportion of the medium and serum is discharged until the unit is abouthalf full. The discs are then rotated at a speed of l revolution/8minutes and air or a mixture of air and 5% CO is passed through the unitat a rate of cc/minute until the cell growth cycle has been completed atwhich time the spent medium is discharged from the propagator, thepropagator is washed with Hanks solution and charged with fresh Medium199 containing 60 ml. 2.8% NaHCO;,/L 25% SPGA and 4 millilitres of amumps virus suspension which has a log,., TClD /0.l ml. of 3.6. Thediscs in the unit are again rotated at a speed of l revolution/8 minutesuntil there is no further increase in the concentration of virus in thesupernatant fluids at which time the vaccine is harvested and frozen.

The yield of mumps vaccine when prepared by the above process issubstantially higher than the yield obtained by using conventionalprocedures.

EXAMPLE 2 A rotating titanium disc propagator is charged with a mixtureof 12 billion trypsinized duck embryo cells in Medium 199 containing 45ml. NaHCO /L and 10% fetal calf serum. The charged propagator is thenlocked into the tumbler and tumbled at 37C. end over end at a speed of lrevolution/l0 minutes until two side plating has occurred at which timethe propagator is removed from the tumbler. The propagator is thenpositioned so that the plane of the discs is in the vertical axis and aportion of the medium and serum is discharged until the unit is abouthalf full. The discs are then rotated at a speed of l revolution/8minutes and air or a mixture of 95% air and 5% CO is passed through theunit at a rate of 100 cc/minute until the cell growth cycle has beencompleted at which time the spent medium is discharged from thepropagator. The propagator is then recharged with 2 litres of Medium 199containing 60 ml. 2.8% NaHCO /L, 2% cit-gamma.

calf serum and ml. of a rubella virus suspension which has log, IND /0.lml. of 3.5. The propagator and contents are then locked into the tumblerand tumbled end over end at a speed of l revolution/ 1 5 minutes for 2hours after which the propagator is removed from the tumbler and afurther 6 litres of Medium 199 containing 60 ml. 2.8% NaHCO /L and 2%a-gamma calf serum is added to the propagator. The unit is then set sothat the discs rotate at one revolution in 8 minutes with air or amixture of air or 95% air and 5% CO passing through it at 100 cc/minuteat 37C. When the infection process has been completed the spent mediumis disharged and fresh Medium 199 containing 60 ml. 2.8% NaHCO /L 10%SPGA is added to the propagator. This medium in turn is discharged whenthe concentration of rubella virus has reached a maximum concentration.

This yield of rubella vaccine when prepared by the above process issubstantially higher than the yield obtained by using conventionalprocedures.

EXAMPLE 3 A rotating titanium disc propagator is charged with a mixtureof Medium 199, F 10 and tryptose phosphate broth with 5% fetal calfserum and 12 X 10 cells from trypsinized 12 day duck embryos and 14.4 X10' PFU Marek THV. The charged propagator is then locked into thetumbler and tumbled at 37C. end over end at a speed of l revolution/20minutes until two side plating has occurred at which time the propagatoris removed from the tumbler. The propagator is then positioned so thatthe plane of the discs is in the vertical axis and a portion of themedium and serum is discharged until the unit is about half full. Thediscs are then rotated at a speed of 1 revolution/8 minutes and air or amixure of 95% air and 5% CO is passed through the propagator at a flowrate of 100 cc/minute. The pH is adjusted from time to time with 7.5%NaHCO 3 so that it remains within the limits of pH 6.8-7.4. Also glucoseis added to the system periodically so that at no time should theglucose concentration to outside the limits of 15-100 mg/lOO ml. On thesixth day after plating the spent medium is discharged and 6 litres ofKCl-citrate/Trypsin is transferred into the propagator. The discs arerotated two complete revolutions and then the KCl-citrate/Trypsinsolution is voided. The propagator is then looked into the tumbler andheld with the plane of the discs in the horizontal axis for 5 minutesafter which it is rotated about its long axis 90 so that the plane ofthe discs is in the vertical axis. While in this position 1.5 L ofEagles Basel Medium containing 15% fetal calf serum is pumped into thepropagator. The propagator and its contents are then rotated end overend at a speed of l revolution/- second for minutes. Following thistumbling the contents are discharged and assayed for Marek THV.

The yield of Mareks vaccine when prepared by the above process issubstantially higher than the yield obtained by using conventionalprocedures.

EXAMPLE 4 A rotating titanium disc propagator is charged with a mixtureof 300 X 10 Wl-38 cells in Medium EBME containing 10% fetal calf serumand 10 ml. of glutamine/L. The propagator and its contents are then heldwith the plane of the plates in the horizontal axis at 37C until platinghas been achieved. The propagator is then positioned so that the planeof the discs is in the vertical axis and a portion of the medium andserum is discharged until the unit is about half full. The discs arethen rotated at a speed of l revolution/5 minutes and air or a mixtureof 5% CO and 95% air is passed through the unit at a rate of 100cc/minute. Twentyfour hours later the medium is discharged from themachine and the unit is refilled with an equal volume of fresh MediumEBME containing 5% fetal calf serum and 10 ml. glutamine/L. After afurther 48 hours in the rotating and gassing mode, the cell suspensionis harvested from the unit. For this operation the unit is voided ofspent medium and is then half filled with a solution containing trypsin.The plates are rotated through the trypsin solution so that all partsof. each plate contact the solution. The trypsin is then voided. Afterwaiting 5 minutes, 1 L of Medium EBME containing fetal calf serum ispumped into the propagator and the whole unit is then tumbled end overend at a speed of l rev./sec. for 10 minutes, after which the cellsuspension is discharged and collected. By use of the above procedurethere is obtained an increase of cell yield of 300% over theconventional procedure.

EXAMPLE 5 A rotating disc propagator is charged with a mixture 6 of 3.0billion trypsinized chick embryo cells, Medium 199, 45 ml. 2.8% NaHCQ-JLand 5% fetal calf serum. The propagator is held in the vertical positionat a temperature of 37C and plating is effected. After 3 hours the fluidin the propagator is discharged and a further 3.0 billion trypsinizedchick embryo cells are added to the fluid. After mixing the freshsuspension is transferred back into the propagator which is held in the0pposite vertical position to the first plating at a temperature of 37Cin order to effect plating on the second side of the discs. When thishas been accomplished, the propagator is positioned so that the plane ofthe discs is in the vertical axis and a portion of the medium and serumis discharged until the unit is about half full. The discs are thenrotated at a speed of l revolution in 5 minutes and air or a mixture of5% CO and air is passed through the propagator ata rate of cc/min. Whenthe cells have reached the confluent state or the growth has ceased, thecells may be harvested.

The medium in the propagator is discharged and the propagator is filledup to the halfway mark with a solution containing trypsin. The discs arerotated twice so that all parts of the disc become wetted with the tryp-The trypsin solution is then discharged and the unit is held with theplane of the plates in the horizontal axis for 5 minutes. The propagatoris then charged with l L of fresh Medium 199 and 45 ml. 2.8% NaHCO /Land is tumbled end over end at a speed of 1 rev./min. for 10 minutesafter which the cell suspension is discharged.

In this way it was possible to prepare with great efficiency and economylarge quantities of chick embryo cells.

EXAMPLE 6 A rotating disc propagator is charged with a mixture of 3.0billion trypsinized duck embryo cells, Medium 199, F 10 5% fetal calfserum and 30 ml. 2.8% NaH- CO /L. The propagator is held in the verticalposition at a temperature of 37C and plating is effected. After threehours the fluid in the propagator is discharged and a further 3.0billion trypsinized duck embryo cells are added to the fluid. Aftermixing the fresh suspension is transferred back into the propagatorwhich is held in the opposite vertical position to the first plating ata temperature of 37C in order to effect plating on the second side ofthe discs. When this has been accomplished, the propagator is positionedso that the plane of the discs is in the vertical axis and a portion ofthe medium and serum is discharged until the unit is about half full.The discs are then rotated at a speed of 1 revolution in 5 minutes andair or a mixture of 5% CO and 95% air is passed through the propagatorat a rate of 100 cc/min. When the cells have reached the confluent stateor the growth has ceased, the cells may be harvested.

The medium in the propagator is discharged and the propagator is filledup to the halfway mark with a solution containing trypsin. The discs arerotated twice so that all parts of the disc become wetted with thetrypsin.

The trypsin solution is then discharged and the unit is held with theplane of the plates in the horizontal axis for 5 minutes. The propagatoris then charged with l L of fresh Medium 199, E10 5% fetal calf serum,30 ml. 2.8% NaHCO lL and is tumbled end over end at a speed of lrev./sec. for 10 minutes after which the cell within the said propagatorand are positioned in suspension is discharged. planes parallel to theend plates of said propagator In this way it was possible to preparewith great effiand are rotatively mounted on a shaft that is posiciencyand economy large quantities of duck embryo tioned in the center of eachof said end plates; cells. 5 means for holding the propagator; and meansfor What is claimed is: tumbling said propagator end over end.

1. A device for use in the production of at least one 2. A device as inclaim 1 which further comprises member selected from the groupconsisting of cells and means to control the tumbling speed of thepropagator. vaccines which comprises;

a multiplate propagator comprising a substantially n 3. A device as inclaim 1 wherein the tumbling means cylindrical vessel having two endplates in which include achain driven sprocket wheel which is engagedthe desired cells and vaccines are grown; by an arm on said means forholding the propagator.

a plurality of spaced apart discs that are located

1. A DEVICE FOR USE IN THE PRODUCTION OF AT LEAST ONE MEMBER SELECTED FROM THE GROUP CONSISTING OF CELLS AND VACCINES WHICH COMPRISES; A MULTIPLATE PROPAGATOR COMPRISING A SUBSTANTIALLY CYLINDRICAL VESSEL HAVING TWO END PLATES IN WHICH THE DESIRED CELLS AND VACCINES ARE GROWN; A PLURALITY OF SPACED APART DISCS THAT ARE LOCATED WITHIN THE SAID PROPAGATOR AND ARE POSITIONED IN PLANES PARALLEL TO THE END PLATES OF SAID PROGATOR AND ARE ROTATIVELY MOUNTED ON A SHAFT THAT IS POSITIONED IN THE CENTER OF EACH OF SAID END PLATES; MEANS FOR HOLDING THE PROPAGATOR; AND MEANS FOR TUMBLING SAID PROPAGOTOR END OVER END.
 2. A device as in claim 1 which further comprises means to control the tumbling speed of the propagator.
 3. A device as in claim 1 wherein the tumbling means include a chain driven sprocket wheel which is engaged by an arm on said means for holding the propagator. 